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PCR Troubleshooting

PCR Troubleshooting. Dave Palmer, Bio-Rad. How PCR works. Cold Spring Harbor Animation PCR.EXE. Review: The structure of DNA. Helix. Complementary Base Pairing. Multiplex PCR: What. PCR using several primer pairs SIMULTANEOUSLY Typically generates a product band for each primer pair.

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PCR Troubleshooting

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  1. PCR Troubleshooting Dave Palmer, Bio-Rad

  2. How PCR works • Cold Spring Harbor Animation PCR.EXE

  3. Review: The structure of DNA Helix Complementary Base Pairing

  4. Multiplex PCR: What • PCR using several primer pairs SIMULTANEOUSLY • Typically generates a product band for each primer pair

  5. Multiplex PCR: Why • Detect several genes at once • eg. transgenic plant screen • Internal controls • VERY important • Tells you how well the PCR reaction worked • Reduces “false negatives” • Reduces “false positives”

  6. Multiplex PCR: How • Same as regular PCR • Care in primer design • Much greater chance of primer-dimers • Much greater chance of artifacts • Annealing temperatures must be close

  7. A Typical PCR Reaction… Sterile Water 38.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTP’s (10mM each) 1.0 ul PrimerFWD (25 pmol/ul) 1.0 ul PrimerREV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul

  8. A Typical Multiplex PCR Reaction Sterile Water 34.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTP’s (10mM each) 1.0 ul Primer1FWD 1.0 ul Primer1REV 1.0 ul Primer2FWD 1.0 ul Primer2REV 1.0 ul Primer3FWD 1.0 ul Primer3REV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul

  9. Multiplex PCR: Example • Three primer pairs • Control, resistance, and trait genes • Control gene fragment is largest and (almost) faintest • Trait gene is smallest and brightest Control Gene Resistance Gene Trait Gene Primers

  10. Multiplex PCR: Example • Three primer pairs Control Gene Resistance Gene Trait Gene Primers Which are transgenic?

  11. Other Types of PCR • Different templates • Nested PCR • RT-PCR (reverse-transcriptase) • Different protocols • iPCR • Touchdown PCR • Real-time PCR

  12. PCR Troubleshooting The effect of each component

  13. PCR Reaction Components • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase • Extras

  14. Water Purity Contamination Amplification Products PCR Reaction Components

  15. Buffer Must match polymerase Typically contain KCl and Tris Can vary over a slight range: Not much difference in range from 0.8 X to 2.0 X Primer efficiency reduced outside this range PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p06.html

  16. DNA template Amount of DNA present Less DNA means more cycles Complexity of DNA Eg. plasmid vs. whole genome Purity Interfering factors, eg. enzymes, salts Degradation PCR more forgiving of degraded DNA Contamination Amplification products Presence of “poisons” Eg. EDTA which scavenges Mg++ PCR Reaction Components

  17. Primers Age Number of freeze-thaws Contamination Amount Can vary over a wide range (50X) 100-500 nM typical Too low: low amplification Too high: low amplification PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p05.html

  18. Nucleotides 20-400 uM works well Too much: can lead to mispriming and errors Too much: can scavenge Mg++ Too low: faint products Age Number of freeze-thaws Just 3-5 cycles is enough to make PCRs not work well Dilute in buffer (eg. 10mM Tris pH 8.0 to prevent acid hydrolysis) Contamination PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p13.html

  19. Mg++ ions Mg is an essential cofactor of DNA polymerase Amount can vary 0.5 to 3.5 uM suggested Too low: Taq won’t work Too high: mispriming PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p14.html

  20. PCR Reaction Components • Bottom Line: • All components work over a wide range. • Need to avoid contamination. • Optimization by trial-and-error.

  21. DNA Polymerase Thermostable? Activity declines with time at 95C Matches buffer? Age Contamination Concentration: Typically 0.5 to 1.0 U/rxn PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p12.html

  22. Extras Proprietary or added by user Glycerol, DMSO Stabilize Taq, decrease secondary structure May help or hurt, depending on primers Typically already in the Taq stock BSA Frequently helps, doesn’t hurt Betaine Useful for GC-rich templates PCR Reaction Components http://info.med.yale.edu/genetics/ward/tavi/p16.html http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html

  23. Denaturation Temp Annealing Temp Extension Temp Time Number of Cycles Reaction Volume “Odd” Protocols PCR Cycling Parameters

  24. Denaturation Step Must balance DNA denaturation with Taq damage 95C for 30 - 60s typically is enough to denature DNA Even 92C for 1s can be enough Taq loses activity at high temps: Half-life at 95C: 40 min Half-life at 97.5C: 5 min PCR Cycling Parameters http://info.med.yale.edu/genetics/ward/tavi/p08.html

  25. Annealing Step Most critical step Calculate based on Tm Often does not give expected results Trial-and-Error Almost always must be done anyway Too hot: no products Too cool: non-specific products Gradient thermocyclers very useful Typically only 20s needed for primers to anneal PCR Cycling Parameters http://info.med.yale.edu/genetics/ward/tavi/p08.html

  26. Extension Step Temperature typically 72C Reaction will also work well at 65C or other temps Time (in minutes) roughly equal to size of the largest product in kb Polymerase runs at 60bp/s under optimum conditions Final “long” extension step mostly unnecessary PCR Cycling Parameters http://info.med.yale.edu/genetics/ward/tavi/p08.html http://info.med.yale.edu/genetics/ward/tavi/p10.html

  27. Number of Cycles Number of source molecules: >100,000: 25-30 >10,000: 30-35 >1,000: 35-40 <50: 20-30 fb. nested PCR Do not run more than 40 Virtually no gain Extremely high chance of non-specific products Best optimized by trial-and-error PCR Cycling Parameters http://info.med.yale.edu/genetics/ward/tavi/p08.html

  28. Reaction Volume Doesn’t affect PCR results as long as volume is within limits. Heated lid important. 5ul, 20ul, 100ul all work. Slightly higher yield with lower volumes. PCR Cycling Parameters http://info.med.yale.edu/genetics/ward/tavi/p03.html

  29. “Odd” Protocols Hot-Start PCR Taq is added last Touchdown PCR Annealing temp is progressively reduced PCR Cycling Parameters

  30. Using right plastic? Using right seals? Heated lid enabled? Right protocol entered? Is power reliable? Proper reaction volume? Programming the Instrument

  31. Basic Experimental Design • A well-designed experiment can keep you from ever getting into trouble! • A poorly-designed experiment is asking for problems!!!!

  32. Basic Experimental Design • Main point: Always use CONTROLS • Positive control • So you’ll know what a successful result looks like. • Negative control • Lets you know if you have contamination.

  33. Experimental Design: Controls No positive or negative controls… What does this result mean?? U Only a positive control… How do we know the result isn’t due to contamination? + U Both positive and negative controls… Results can be interpreted with confidence. U - +

  34. Experimental Design: Replication Our unknown is definitely positive... …but how sure are we? - + We ran the same sample three times…. Is our unknown really positive? - + U U U

  35. Review Here are the key things to know…

  36. Review: A Typical PCR Reaction Sterile Water 38.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTP’s (10mM each) 1.0 ul PrimerFWD (25 pmol/ul) 1.0 ul PrimerREV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul

  37. Review: Purpose of Temp Cycling • Denaturing • Annealing • Extension

  38. Review: Practical uses of PCR • Disease detection • Cloning • Forensics • Food quality control • Paternity testing • Identification

  39. Review: Basic Experimental Design • Unknown Samples • Controls • Positive Controls • Negative controls • Replication!

  40. End of PCR Troubleshooting

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